Photolytic Cross-Linking to Probe Protein–Protein and Protein–Matrix Interactions in Lyophilized Powders

Abstract

Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic cross-linking with mass spectrometric analysis (ssPC–MS). In order to characterize structure and microenvironment, protein–protein, protein–excipient, and protein–water interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4′-azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography/mass spectrometry (LC–MS). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce cross-linked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to five labels, as detected by LC–MS. Following lyophilization and irradiation, cross-linked peptide–peptide, peptide–water, and peptide–raffinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide–peptide, peptide–water, and peptide–excipient adducts detected. In the absence of excipients, peptide–peptide adducts involving the CD, DE, and EF loops and helix H were common. In the raffinose formulation, peptide–peptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein–protein and protein–water adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC–MS can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution